水母的寿命
目录
中文摘要 (1)
英文摘要 (3)
英文缩写 (5)
研究论文骨髓间充质干细胞来源外泌体对酸性环境下人髓核细胞的保护性研究快速抑制呕吐感
前言 (6)
材料与方法 (8)
结果 (15)
讨论 (23)
结论 (25)
参考文献 (25)
综述椎间盘退变的研究进展 (30)
工作展望简短致谢 (43)
个人简历 (44)
骨髓间充质干细胞来源外泌体对酸性环境下人髓核细胞的保护
性研究
童年无忌
摘要
目的：探索骨髓间充质干细胞来源的外泌体对酸性环境下人椎间盘髓核细胞的保护作用。
方法：
1．观察人椎间盘髓核细胞在不同酸性培养基中（A组pH（7.1-7.3），B组pH（6.5-6.7）C组pH（5.9-6.1））细胞凋亡蛋白Caspa-3、细胞外基质蛋白Aggrecan及Collagen II以及细胞外基质降解相关蛋白MMP-13表达水平。
2. 骨髓间充质干细胞培养密度达70%时更换无血清培养基,48h后收集条件培养基用于外泌体提取。使
用Nanosight或透射电镜分析外泌体的大小及形态。使用Western 印迹法检测外泌体标志物TSG101和CD63的表达。
3. 探索(D组pH（7.1-7.3），E组pH（5.9-6.1）,F组pH（5.9-6.1）+Exo)酸性环境中入外泌体后髓核细胞凋亡蛋白Caspa-3、细胞外基质蛋白Aggrecan、Collagen II以及基质金属蛋白酶MMP-13表达量的改变。
尊重他人的作文
结果：
1. 凋亡蛋白Caspa-3，基质金属蛋白酶MMP-13在B、C组酸性培养基中表达升高，与A组相比差异具有统计学意义（p＜0.05）。细胞外基质相关蛋白Aggrecan在B、C组酸性培养基中表达降低，与A组相比差异具有统计学意义（p＜0.05），Collagen II在C组酸性条件下表达降低，与B组相比具有统计学意义（p＜0.05）。糕点制作
2. 通过差速离心后获得沉淀物在投射电镜下观察到直径约为100nm 的茶托样形态，粒径分析显示沉淀物直径峰值约为125nm，Western印迹法检测发现收集的沉淀物表达外泌体特异性抗体TSG101和CD63，表明成功从骨髓间充质干细胞中提取到外泌体。
老银匠银饰加盟3. 当pH为5.9~6.1酸性培养基中外泌体终浓度达到20μM时，Western蛋白印迹法检测显示Caspa-3及MMP-13表达降低，而
Aggrecan及Collagen II表达增加。
结论：
在低pH（5.9-6.1）条件下可以诱导髓核细胞细胞凋亡蛋白Caspa-3及金属蛋白酶MMP-13表达增加，胞外基质Aggrecan、Collagen II分泌减少，而骨髓间充质干细胞来源外泌体能够对此酸性环境下髓核细胞产生保护作用。此项研究为治疗椎间盘退变提供潜在的治疗策略。
关键词：骨髓间充质干细胞，外泌体，髓核细胞，腰椎退行性变
Exosome derived from bone marrow menchymal stem cells protects human nucleus pulposus cells from acidic pH-induced
damage
ABSTRACT
Objective:To explore the protective of exosome on nucleus pulp osus cell in acidic pH.键盘多少钱
Methods:
1．NPC were divided into three groups: Group A, pH 7.1~7.3; Group B, pH 6.5~6.7 and Group C, pH 5.9~6.1. The NPCs were cultured in above-defined acidic medium, simultaneously, three different amounts of Exo were added into the media. Finally, the expression of the caspa-3, aggrecan, collagen II and MMP-13 was analyzed and compared among the different groups.
2. Bone menchymal stem cell culture medium was replaced with exosome-free medium before the culture reached 80% confluence. After 48 h, the cultured supernatant was collected for exosome isolation. The number, size distribution and morphological of the isolated particles were analyzed using the Nanoparticle Tracking Analyzer PMX110 and transmission electron microscope. Western blot analysis was performed to detect the expression of Exo markers (TSG101, CD63).
3. Finally, NPCs were cultured in the above-defined acidic medium supplemented with appropriate concentration of BMSCs-Exo. The expression of caspa-3, MMP-13, aggrecan and collagen II was detected by Western blot and qRT-PCR.
Results: 1.The results of western blot analysis showed that the expression of cleaved caspa-3 and MMP-13 in Group B and C was significantly incread compared with group A (p<0.05). However, the expression of collagen II was significantly decread in Group C compared with B (p<0.05), and the expression of aggrecan was also
decread significantly in Group B and C compared with A (p<0.05)；2. The morphology of BMSC-derived Exo appeared as cup-shaped vesicles with a size of about 100 nm, as obrved by transmission electron microscopy. Particle size and distribution of the isolated Exo were analyzed by the Nanoparticle Tracking Analyzer and produced a absorption peak in the region of about 125 nm. The expression of the known exosomal markers TSG101 and CD63 was confirmed by Western blot analysis. The above results identified the particles collected from MSC culture medium as BMSC-Exo. 3. When BMSC-derived Exo equivalent to 20 μM of BMSC-Exo protein was added into NPCs cultured at pH 5.9-6.1 medium, the upregulation of cleaved caspa-3 and MMP-13 as well as the downregulation of collagen II and aggrecan induced by acidic pH were significantly reverd, as shown by Western blot analysis.
Conclusions:
In the pathological acid environment, MSC-derived Exo promotes the expression of chondrocyte extracellular matrix, collagen II and aggrecan, and reduces matrix degradation by downregulating matrix degrading enzymes, protecting NPCs from acidic pH-induced apoptosis. This study reveals a potential promising strategy for treatment of IVD degeneration.
Keywords: Bone menchymal stem cell, Exosome, Lumbar disc degeneration, Acidic medium.
